微生物限度译文usp

1、微生物限度检验微生物限度检验本章提供了从原材料到成品的试验，包括需氧微生物数量的预测和所有药物条款中规定的微生物种类的预防试验。假如自动化的方法经验证和现在行方法等效或有更好的结果，可以取代现在的试验。在准备和实验中，处理样品时遵守无菌操作。除非有特别说明，样品简单“培养”的地方，应把容器放在 30-35的空气中恒温培养 2448 小时。术语“生长”经常用在此处，换言之，指明微生物的存在和预测增殖。预备试验预备试验在本章实验中实验结果的验证提供了大量充分的论证，用于试验的样品本身在实验条件下不限制可能存在的微生物的生长。因而，用固定成分做试验的预备和环境的需求，接着分别用金黄色葡萄球菌，大肠埃

2、希菌，铜绿假单孢菌和沙门菌接种检验材料的稀释样品中。可以通过加 1 毫升不少于 10浓度的肉汤培养 24 小时的微生物的稀释液到实验材料的第一步稀释液（PH7.2 的磷酸盐缓冲液，大豆酪蛋白消化物培养基或乳糖液状培养基）中和下面的实验过程来实现。在有关培养基中没有微生物生长，检验内容无效，需要变更程序，包括（1）对剩余的相同数量的实验材料增大稀释液的体积，或（2）在稀释液中掺入适宜的灭活剂，或（3）通过（1） （2）的共同作用，使接种物能够生长。下面是一个配料和他们的聚合物的例子，他们可以加到培养基中灭活样品中的抑菌物质：大豆蛋黄素（卵磷脂） 。0.5%；聚山梨脂 20，4.0%。作为比较，重

3、复上面描述的实验，用液体酪蛋白消化物-大豆蛋黄素聚山梨脂 20 培养基来验证实验材料中存在的防腐剂或其他抑制剂的灭活作用。如果抑菌物质存在样品中并且是可溶的，适宜的经验证的程序可以用于薄膜过滤技术（样品的无菌实验）中。如果加了适量的灭火剂和增大了稀释液体积，仍不能恢复上面描述和不适用于薄膜过滤技术中物品的培养基的活力，能假定分离接种微生物的失败原因可归于产品的杀菌活力。该信息用于显示物品不可能被给定的微生物种类污染。为了确立物品的抑菌谱和杀菌活力，应继续监测。缓冲液和培养基缓冲液和培养基培养基可以按下面的准备，或者假若脱水培养基是有供货商或分销商制备，可以被应用，他们与给定比例获得的培养基想比

4、有相似的成分。用给定的比例成分制备培养基，在水中溶解可溶性固体物，如果需要，加热溶解完全，使用的时候加盐酸或氢氧化钠到溶液中是培养基中的 PH 适宜。在 252测定 PH 值。配方中含琼脂时，使用含水量不超过 15%的琼脂。配方中需用水时，使用纯水。PH7.2 盐酸缓冲液原液在 1000 毫升容量瓶中用 500 毫升水溶解 34 克磷酸二氢钾，加氢氧化钠（约175 毫升）调 PH 到 7.20.1，加水到体积线，混匀。分装后灭菌。冷藏储存。用的时候，用水按 1：800 稀释原液，灭菌。培养基除非有其他说明，培养基应该在高压蒸汽灭菌器（见无菌中的蒸汽灭菌法）内加热灭菌，灭菌时间要参考灭菌体积。1

5、，液体酪蛋白-大豆蛋黄素-吐温 20 培养基酪蛋白胰酶消化物 20 g大豆蛋黄素5 g吐温 2040 mL水960 mL在 960 毫升水中溶解酪蛋白胰酶消化物和大豆蛋黄素，在 48-50的水浴中加热 30分钟使溶解完全。加 40 毫升吐温 20，混匀，按需要的分装。 2，大豆蛋黄素消化物琼脂培养基酪蛋白胰酶消化物15.0 g大豆粉木瓜酶消化物 5.0 g氯化钠5.0 g琼脂15.0 g水1000 mL 灭菌后 PH：7.30.2。3，液体大豆蛋黄素培养基 参照无菌检测下大豆蛋黄素消化物培养基的配制。4，甘露醇盐琼脂培养基酪蛋白胰酶消化物5.0 g动物组织的胃蛋白酶的消化物 5.0 g牛肉膏1

6、.0 gD-甘露醇10.0 g氯化钠75.0 g琼脂15.0 g酚磺酞0.025 g水1000 mL 混匀，加热沸腾 1 分钟，并快速搅拌使溶解。 灭菌后 PH 值：7.40.2。5， BairdParker 琼脂培养基酪蛋白胰酶消化物 10.0 g牛肉膏5.0 g酵母膏1.0 g氯化锂5.0 g琼脂20.0 g甘氨酸12.0 g丙酮酸钠10.0 g水950 mL加热并快速搅拌，沸腾 1 分钟。灭菌，冷却到 4050，加入 10 毫升亚碲酸钾溶液（1：100）和 50 毫升蛋黄乳液。轻摇混匀，倒入盘子中。 （蛋黄乳液：对整个鸡蛋外壳消毒，无菌打开鸡蛋，把蛋黄分离到一无菌量筒中。加无菌盐水 TS

7、，得到一个 3：7 的盐水蛋黄液。加到一无菌搅拌器中，高速混匀 5 秒钟。 ）灭菌后 PH 值：6.80.2。6，VogelJohnson 琼脂培养基 酪蛋白胰酶消化物 10.0 g酵母膏5.0 g甘露醇10.0 g磷酸氢二钾5.0 g氯化锂5.0 g甘氨酸10.0 g琼脂16.0 g酚磺酞25.0 mg水1000 mL煮沸固体溶液 1 分钟。 灭菌，冷却到 4550，加入 20 毫升无菌亚碲酸钾溶液（1：100）。灭菌后 PH 值： 7.2 0.2.7， 溴棕三甲铵琼脂培养基 胰脏明胶消化物20.0 g氯化镁1.4 g硫酸钾10.0 g琼脂13.6 g溴化十六烷基三甲铵 0.3 g丙三醇10

8、.0 mL水1000 mL在水中溶解固体组分，加丙三醇。加热沸腾 1 分钟，并快速搅拌使溶解。灭菌后 PH 值： 7.2 0.2.8，假单胞菌属琼脂培养基（用于氟化荧光素的检出） 酪蛋白胰酶消化物10.0 g动物组织的胃蛋白酶消化物 10.0 g无水磷酸氢二钾1.5 g硫酸镁 (MgSO47H2O)1.5 g丙三醇10.0 mL琼脂15.0 g水1000 mL在水中溶解固体组分，加丙三醇。加热沸腾 1 分钟，并快速搅拌使溶解。灭菌后 PH 值： 7.2 0.2.9，假单胞菌属琼脂培养基（用于绿脓菌素的检出）胰脏明胶消化物 20.0 g无水氯化镁1.4 g无水硫酸钾10.0 g琼脂15.0 g丙

9、三醇10.0 mL水1000 mL在水中溶解固体组分，加丙三醇。加热沸腾 1 分钟，并快速搅拌使溶解。灭菌后 PH 值： 7.2 0.2。.10，乳糖液状培养基牛肉膏3.0 g胰脏明胶消化物 5.0 g乳糖5.0 g水1000 mL灭菌后尽可能快的冷却。灭菌后 PH 值：6.9 0.2.11，流体亚硒酸盐-胱氨酸培养基 酪蛋白胰酶消化物 5.0 g乳糖4.0 g磷酸钠10.0 g亚硒酸钠4.0 gL-胱氨酸10.0 mg水1000 mL最终 pH: 7.0 0.2.混匀，加热溶解。在流动的蒸汽中加热 15 分钟，不可以灭菌不可以灭菌。12，液体 Tetrathionate 培养基酪蛋白胰酶消化

10、物2.5 g动物组织的胃蛋白酶消化物 2.5 g胆（汁）盐1.0 g碳酸钙10.0 g硫代硫酸钠30.0 g水1000 mL加热固体溶液至沸腾。在使用的时候，加入配制好的碘液 20 毫升（在 20 毫升水中溶解 5克碘化钾和 6 克碘）。接着加入 10 毫升亮绿溶液（1：1000），混匀。加入亮绿溶液后不可以加热培养基。13，亮绿琼脂培养基 酵母膏3.0 g动物组织的胃蛋白酶消化物 5.0 g酪蛋白胰酶消化物5.0 g乳糖10.0 g氯化钠5.0 g蔗糖10.0 g酚磺酞80 mg琼脂20.0 g碱性亮绿12.5 mg水1000 mL煮沸固体溶液 1 分钟。临用前灭菌，融化培养基，倒入培养皿中

11、，允许冷却。灭菌后 PH 值： 6.9 0.2.14,木糖赖氨酸去氧胆酸盐琼脂培养基 木糖3.5 gL-赖氨酸5.0 g乳糖7.5 g蔗糖7.5 g氯化钠5.0 g酵母膏3.0 g酚磺酞80 mg琼脂13.5 g去氧胆酸钠 2.5 g硫代硫酸钠 6.8 g枸橼酸铁胺 800 mg水1000 mL最终：pH: 7.4 0.2.回旋摇动加热固体和水的混合物到液体沸腾。不可以过热或灭菌 。立即转入一水浴容器中维持水温在 50 左右， 培养基一冷却就到入培养皿中。15,亚硫酸铋琼脂培养基 牛肉膏5.0 g酪蛋白胰酶消化物5.0 g动物组织的胃蛋白酶消化物 5.0 g葡萄糖5.0 g磷酸钠4.0 g硫酸

12、亚铁300 mg亚硫酸铋指示剂8.0 g琼脂20.0 g碱性亮绿25 mg水1000 mL最终 pH: 7.6 0.2.回旋摇动加热固体和水的混合物到液体沸腾。不可以过热或灭菌 。立即转入一水浴容器中维持水温在 50 左右， 培养基一冷却就到入培养皿中。16，三糖-铁-琼脂培养基 酪蛋白胰酶消化物10.0 g动物组织的胰酶消化物 10.0 g乳糖10.0 g蔗糖10.0 g葡萄糖1.0 g硫酸亚铁铵200 mg氯化钠5.0 g硫代硫酸钠200 mg琼脂13.0 g酚磺酞25 mg水1000 mL灭菌后 PH： 7.3 0.2.17，麦康基琼脂培养基 动物胶胰酶消化物17.0 g酪蛋白胰酶消化物

13、1.5 g动物组织胃蛋白酶消化物 1.5 g乳糖10.0 g胆盐混合物1.5 g氯化钠5.0 g琼脂13.5 g中性红30 mg结晶紫1.0 mg水1000 mL加热固体和水的混合物沸腾 1 分钟使溶解。灭菌后 PH：7.1 0.2.18，肠杆菌科曙红亚甲蓝琼脂培养基 动物胶胰酶消化物 10.0 g磷酸氢二钾2.0 g琼脂15.0 g乳糖10.0 g曙红 Y400 mg亚甲蓝65 mg水1000 mL在水中溶解动物胶胰酶消化物，磷酸氢二钾和琼脂，可以冷却。 仅仅在使用前，熔解胶状琼脂培养基，熔解时按下面的分量加入剩余的组分并混匀： 每 100 毫升胶状琼脂培养基5 mL 乳糖溶液 (1：5),

14、 2 mL 曙红 Y 溶液 (1：50), 和 2 mL 亚甲蓝溶液 (1：300)。 配制好的培养基允许不澄清。灭菌后 PH： 7.1 0.2.19，萨布罗右旋琼脂培养基 葡萄糖40 g动物组织胰酶消化物和酪蛋白胰酶消化物的等量混合物 10 g琼脂15 g水1000 mL混匀，加热沸腾使溶解。灭菌后 PH：5.6 0.2.20，马铃薯右旋琼脂培养基 在 500 毫升蒸馏水中煮 300 克去皮切成丁的马铃薯,用滤布过滤，加蒸馏水至 1000 毫升，然后加入下面的成分：琼脂15 g葡萄糖20 g加热溶解，灭菌。灭菌后 PH： 5.6 0.2.使用时，在倒培养皿前，用酒石酸溶液（1：10）调整熔解

15、冷却到 45 的培养基至 PH 3.5 0.1.。不可以再加热 pH 3.5 的培养基。取样取样按论文中要求的预备 10-mL or 10-gs 样品用于实验。操作过程操作过程 通过与样品物理特性相适应的方法准备待测样品，不能改变样品中本来存在的微生物的种类和数量，获得一个溶液，或全部的悬浮液，或一个适于实验过程的形状的部分。对于一个在可察觉的范围内溶解但不完全溶解的固体，把这个固体变成适度的细粉状，在特定的媒介物中做成悬浮液，按下面所示的程序进行：总的需氧微生物计数，金黄色葡萄球菌实验，铜绿假单胞菌实验，沙门菌和大肠杆菌实验。对于液体样品，包括真溶液，水中的悬浮液或含水酒精（酒精含量不少于

16、30%）；和在90 毫升 PH7.2 的磷酸盐缓冲液中能快速并完全溶解的固体样品，按下面所示的程序进行：总的需氧微生物计数，金黄色葡萄球菌实验，铜绿假单胞菌实验，沙门菌和大肠杆菌实验。For water-immiscible fluids, ointments, creams, and waxes, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying agent (such as one of the polysorbates), using a mechanica

17、l blender and warming to a temperature not exceeding 45 , if necessary, and proceed with the suspension as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.For a fluid specimen in ae

18、rosol form, chill the container in an alcohol-dry ice mixture for approximately 1 hour, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quantity of test material required for the procedures

19、 specified in one of the two preceding paragraphs, as appropriate. Where 10.0 g or 10.0 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape,

20、and proceed with the test on the residues. If the results of the test are inconclusive or doubtful, repeat the test with a specimen from 20 more containers.Total Aerobic Microbial Count For specimens that are sufficiently soluble or translucent to permit use of the Plate Method, use that method; oth

21、erwise, use the Multiple-Tube Method. With either method, first dissolve or suspend 10.0 g of the specimen if it is a solid, or 10 mL, accurately measured, if the specimen is a liquid, in pH 7.2 Phosphate Buffer, Fluid SoybeanCasein Digest Medium, or Fluid Casein DigestSoy Lecithin-Polysorbate 20 Me

22、dium to make 100 mL. For viscous specimens that cannot be pipeted at this initial 1:10 dilution, dilute the specimen until a suspension is obtained, i.e., 1:50 or 1:100, etc., that can be pipeted. Perform the test for absence of inhibitory (antimicrobial) properties as described under Preparatory Te

23、sting before the determination of Total Aerobic Microbial Count. Add the specimen to the medium not more than 1 hour after preparing the appropriate dilutions for inoculation.PLATE METHOD Dilute further, if necessary, the fluid so that 1 mL will be expected to yield between 30 and 300 colonies. Pipe

24、t 1 mL of the final dilution onto each of two sterile petri dishes. Promptly add to each dish 15 to 20 mL of SoybeanCasein Digest Agar Medium that previously has been melted and cooled to approximately 45 . Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and a

25、llow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 48 to 72 hours. Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per g or per mL of speci

26、men. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as “less than 10 microorganisms per g or per mL of specimen.”MULTIPLE-TUBE METHOD Into each of fourteen test tubes of similar size place 9.0 mL of sterile Fluid Soy

27、beanCasein Digest Medium. Arrange twelve of the tubes in four sets of three tubes each. Put aside one set of three tubes to serve as the controls. Into each of three tubes of one set (“100”) and into a fourth tube (A) pipet 1 mL of the solution or suspension of the specimen, and mix. From tube A, pi

28、pet 1 mL of its contents into the one remaining tube (B) not included in a set, and mix. These two tubes contain 100 mg (or 100 L) and 10 mg (or 10 L) of the specimen, respectively. Into each of the second set (“10”) of three tubes pipet 1 mL from tube A, and into each tube of the third set (“1”) pi

29、pet 1 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the tubes. Following the incubation period, examine the tubes for growth: the three control tubes remain clear and the observations in the tubes containing the specimen, when interpreted by reference

30、to Table 1, indicate the most probable number of microorganisms per g or per mL of specimen.Table 1. Most Probable Total Count by Multiple-Tube Method Observed Combinations of Numbers of Tubes Showing Growth in Each SetNo. of mg (or mL) of Specimen per Tube100(100 L)10 (10 L)1(1 L)Most ProbableNumbe

31、r ofMicroorgan-isms per g or per mL3331100 33211003315003302003232903222103211503209031316031212031170310403039530260Observed Combinations of Numbers of Tubes Showing Growth in Each SetMost ProbableNumber ofMicroorgan-isms per g or per mLNo. of mg (or mL) of Specimen per Tube100(100 L)10 (10 L)1(1 L

32、)3014030023Test for Staphylococcus aureus and Pseudomonas aeruginosa To the specimen add Fluid SoybeanCasein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of VogelJohn

33、son Agar Medium (or BairdParker Agar Medium, or MannitolSalt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and incubate. If, upon examination, none of the plates contains colonies having the characteristics listed in Tables 2 and 3 for the media

34、 used, the test specimen meets the requirements for freedom from Staphylococcus aureus and Pseudomonas aeruginosa. Table 2. Morphologic Characteristics of Staphylococcus aureus on Selective Agar Media Selective MediumCharacteristic Colonial MorphologyGram StainVogel-JohnsonAgar MediumBlack Surrounde

35、d by yellow zonePositive cocci(in clusters)Mannital-SaltAgar MediumYellow colonies with yellow zonesPositive cocci (in clusters)Baird-ParkerAgar MediumBlack, shiny, surrounded by clear zones 2 to 5 mmPositive cocci (in clusters)Table 3. Morphologic Characteristics of Pseudomonas aeruginosa on Select

36、ive and Diagnostic Agar Media Selective MediumCharacteristic Colonial MorphologyFluorescence in UV LightOxidase TestGram StainCentrimide Agar MediumGenerally greenishGreenishPositiveNegative rodsPseudomonas Agar Medium for Detection of FluorescinGenerally colorless to yellowishYellowishPositiveNegat

37、ive rodsPseudomonas Agar Medium forDetection of PyocyaninGenerally greenishBluePositiveNegative rodsCoagulase Test (for Staphylococcus aureus) With the aid of an inoculating loop, transfer representative suspect colonies from the agar surfaces of the VogelJohnson Agar Medium (or BairdParker Agar Med

38、ium, or MannitolSalt Agar Medium) to individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37 , examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and

39、negative controls simultaneously with the unknown specimens. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus. Oxidase and Pigment Tests (for Pseudomonas aeruginosa) With the aid of an inoculating loop, streak represent

40、ative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred, divide the surface of each p

41、late into quadrants, each of which may be inoculated from a separate colony. Cover and invert the inoculated media, and incubate at 35 2 for not less than three days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies having the characteristics listed in T

42、able 3 are present. Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine

43、 dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.Test for

44、 Salmonella species and Escherichia coli To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL, and incubate. Examine the medium for growth, and if growth is present, mix by gently shaking. Pipet 1-mL portions into vessels containing, respectively, 10 m

45、L of Fluid SeleniteCystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 12 to 24 hours. (Retain the remainder of the Fluid Lactose Medium.)Test for Salmonella Species By means of an inoculating loop, streak portions from both the selenite-cystine and tetrathionate media on the surfac

46、e of Brilliant Green Agar Medium, XyloseLysineDesoxycholate Agar Medium, and Bismuth Sulfite Agar Medium contained in petri dishes. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 4, the specimen meets the requirements o

47、f the test for absence of the genus Salmonella. Table 4. Morphologic Characteristics of Salmonella Species on Selective Agar Media Selective MediumCharacteristic Colonial MorphologyBrilliant GreenAgar MediumSmall, transparent, colorless or pink to white opaque (frequently surrounded by pink to red z

48、one)Xylose-Lysine-DesoxycholateAgar MediumRed, with or without black centersSelective MediumCharacteristic Colonial MorphologyBismuth SulfiteAgar MediumBlack or greenIf colonies of Gram-negative rods matching the description in Table 4 are found, proceed with further identification by transferring r

49、epresentative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple SugarIronAgar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface. Incubate. If examination discloses no evidence of tubes having alkaline (

50、red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirements of the test for the absence of the genus Salmonella.* Test for Escherichia coli By means of an inoculating loop, streak a portion from the re

51、maining Fluid Lactose Medium on the surface of MacConkey Agar Medium. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 5 for this medium, the specimen meets the requirements of the test for absence of Escherichia coli. Table 5. Morphologic Characteristics of Escherichia coli on MacConkey Agar Medium Gram StainCharacteristic Colonial MorphologyNegative rods(cocco-bacilli)Brick-red; may have surrounding zone of precipitated bileIf colonies matching the descri